ABSTRACT: Emerging infectious diseases cause population declines in many ectotherms, with outbreaks frequently punctuated by periods of mass mortality. It remains unclear, however, whether thermoregulation by ectotherms and variation in environmental temperature is associated with mortality risk and disease progression, especially in wild populations. Here, we examined environmental and body temperatures of free-ranging eastern box turtles Terrapene carolina during a mass die-off coincident with upper respiratory disease. We recorded deaths of 17 turtles that showed clinical signs of upper respiratory disease among 76 adult turtles encountered in Berea, Kentucky (USA), in 2014. Of the 17 mortalities, 11 occurred approximately 14 d after mean environmental temperature dropped 2.5 SD below the 3 mo mean. Partial genomic sequencing of the major capsid protein from 1 sick turtle identified a ranavirus isolate similar to frog virus 3. Turtles that lacked clinical signs of disease had significantly higher body temperatures (23°C) than sick turtles (21°C) during the mass mortality, but sick turtles that survived and recovered eventually warmed (measured by temperature loggers). Finally, there was a significant negative effect of daily environmental temperature deviation from the 3 mo mean on survival, suggesting that rapid decreases in environmental temperature were correlated with mortality. Our results point to a potential role for environmental temperature variation and body temperature in disease progression and mortality risk of eastern box turtles affected by upper respiratory disease. Given our findings, it is possible that colder or more variable environmental temperatures and an inability to effectively thermoregulate are associated with poorer disease outcomes in eastern box turtles.